Psoralea corylifolia L. (Fabaceae) is a widely used medical plant in China. This study was designed to screen and identify bioactive compounds with anticancer activity from the seeds of Psoralea corylifolia L. One volatile fraction (fraction I) and three other fractions (fraction II, III, IV) from methanol extraction of P. corylifolia L. were obtained. Bioactivities of these fractions were evaluated by the cytotoxicity on KB, KBv200, K562, K562/ADM cancer cells with MTT assay. Major components in the active fraction were identified by HPLC/MS(n). Fraction IV significantly inhibits the growth of cancer cells in a dose-dependent manner. The IC(50) values were 21.6, 24.4, 10.0 and 26.9, respectively. Psoralen and isopsoralen, isolated from fraction IV, were subject to bioactive assay and presented a dose-dependent anticancer activity in four cancer cell lines (KB, KBv200, K562 and K562/ADM). The IC(50) values of psoralen were 88.1, 86.6, 24.4 and 62.6, which of isopsoralen were 61.9, 49.4, 49.6 and 72.0, respectively. Apoptosis of tumor cell significantly increased after treated with psoralen and isopsoralen. Induction of apoptotic activity was confirmed by flow cytometry after staining with Annexin V/PI. These results suggested psoralen and isopsoralen contribute to anticancer effect of P. corylifolia L.